Abstract:[Background] Vibrio parahaemolyticus is a seafood-borne pathogen. CalR is a global transcriptional regulator in V. parahaemolyticus. Type 3 secretion systems 2 (T3SS2) is one of the major virulence determinants of V. parahaemolyticus. VopB2 is a key effector protein of T3SS2. [Objective] To study the transcriptional regulation of vopB2 by CalR in Vibrio parahaemolyticus. [Methods] Primer extension assay was employed to detect the transcription start sites and the amount of primer extension porroducts of target genes in ΔcalR and WT. Quantitative RT-PCR was then carried out to calculate the transcriptional variation of target genes between ΔcalR and WT. LacZ assay was used to verify regulation relationship by measuring the β-galactosidase activities in cellular extracts using a β-Galactosidase Enzyme Assay System (Promega). Finally the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-CalR to target promoters in vitro. [Results] We detected one transcription start site for vopB2, which was located at 130 bp and 28 bp upstream of vopB2 and its transcribed activity was under the negative control of the CalR. We also found that His-CalR was bind to the promoter region of vopB2 directly. Besides, our data showed that CalR had no regulatory effect on vtrA transcription, a known regulator for vopB2. [Conclusion] V. parahaemolyticus CalR represses the transcription of vopB2 directly, which is not associated to vtrA.
